Maintenance of vascular integrity is an important host response to injury. Complex hemostatic mechanisms of coagulation, platelet function, and fibrinolysis exist to minimize adverse consequences of vascular injury and to accelerate vascular repair. Many of these hemostatic mechanisms are initiated and/or regulated by cells of the wall of the blood vessel.
A number of plasma proteins and circulating factors in blood are inactive until the events that stimulate clotting are triggered, at which point the various factors involved in clot formation are activated. Several different types of activation are to be considered in coagulation. One type is the activation of a zymogen of a proteolytic enzyme, the product of which, in turn, activates another zymogen by proteolytic action. Another type does not lead to activation of an enzyme. For example, the proteolytic cleavage of a soluble plasma protein, fibrinogen, results in formation of fibrin, the insoluble protein that forms the clot. Similarly, platelets, the cells that are essential for normal coagulation, must also be activated before they can participate in coagulation.
Classically, the initiation of blood clotting can be conceptually separated into two different, yet similar, molecular mechanisms, called the intrinsic and extrinsic coagulation pathways or cascades. The mechanism of initiation pertaining to this patent application is the extrinsic coagulation pathway. The two pathways converge at a step, the activation of prothrombin to thrombin by factor X.sub.a, prior to the actual clot formation. There is also some feedback regulation between the two systems. In the extrinsic pathway, for optimal conditions, each reaction of the coagulation mechanism involves assembly on a cellular surface of a complex that includes a serine protease, a substrate zymogen, and a nonenzymatic cofactor protein, with or without calcium ions. Complex formation on the cell surfaces localizes the hemostatic response, promotes optimal activation of coagulation, and protects proteases from their plasma inhibitors.
The actual set of cascade reactions of the extrinsic pathway are as follows: in the presence of calcium, found in circulation, tissue factor, a membrane-bound procoagulant enzyme, combines with factor VII, a circulating proenzyme, and the complex is activated to TF-VII.sub.a by either X.sub.a, which can be found in very low concentrations in the circulating blood, or by high molecular weight kallikrein acting on XII.sub.a of the intrinsic pathway, also found in the circulation. Once the complex has been activated it can active X, a proenzyme in blood, to X.sub.a, a serine protease. X.sub.a can now activate, in the presence of factor V, (a cofactor found in circulation), prothrombin to thrombin. Thrombin will then cleave fibrinogen to form fibrin.
Factor V and tissue factor can be synthesized by vascular endothelium, whereas endothelial cells posses binding sites for the plasma proteins, and high molecular weight kininogen, allowing expression of these coagulant activities.
Once thrombin is generated, fibrinogen is cleaved to form fibrin, which is substantially cross-linked by factor XIII.sub.a to form an insoluble fibrin clot. Enmeshed in the clot are platelets that have been recruited to the site of vascular injury after exposure to subendothelial components such as collagen.
When vascular cells are exposed to perturbing stimuli, a number of cellular hemostatic properties are altered, including increased expression of procoagulant activity, decreased expression of anticoagulant activity, and enhanced platelet adhesion and activation.
The coagulation mechanisms can be initiated either by expression of tissue factor activity, (extrinsic pathway), or by activation of factor XII, (intrinsic pathway).
A number of stimuli have been found to induce tissue factor activity. These stimuli include immune modulators such as immune complexes, the monokines interleukin 1 and tumor necrosis factor, other stimuli linked to infection such as endotoxin and microorganisms associated with bacterial endocarditis, a metabolite associated with thrombotic disease (homocysteine), and other stimuli such as mechanical injury and phorbol esters. With the exception of endothelial cell injury induced by mechanical means or by gross infection (such as endocarditis), the induction of these vascular coagulant properties is not normally associated with overt cell injury.